Method for preparing cell populations with anti-tumor immune response activity

ABSTRACT

The invention provides a method for preparing cell populations with anti-tumor immune response activity, which includes co-culturing tumor and mononuclear cell in a three-dimensional cell culture device, separating and amplifying the cell populations with anti-tumor immune response activity from the cultures. The present invention, at the same time, discloses the cell populations with anti-tumor immune response activity obtained by the method and the kit comprising the cell populations.

The present application is a U.S. national stage filing under 35 USC 371of PCT/CN06/03136 filed Nov. 22, 2006.

TECHNICAL FIELD

The present invention belongs to the fields of biotechnology andimmunology, and involves a method for preparing a group of cellpopulations with an anti-tumor immune response activity, and thesensitized cell populations with anti-tumor immune response activityprepared by said method.

BACKGROUND ART

Malignant tumors have become one of the main diseases that threatenhuman health and survival. The conventional therapies of tumor such assurgical operation, radiation therapy and chemotherapy involve externalforces, which include exscinding tumors directly, killing tumor cellswith radiation, or by chemotherapeutics. Chemotherapy and radiationtherapy usually are unable to resolve the issues like tumor metastasisand recurrence. Moreover, these treatments always have severe toxiceffects and damage the normal cells. In particular, radiation therapyand chemotherapy will damage the immune system, especially cell-mediatedimmunity which plays an important role in the body's anti-tumor defense.Increasing attention has been paid to biological treatments which arebased on immunotherapy and promoted by the developments of modernoncology, molecular biology, theoretical immunology, biotechnology, andgenetic engineering technology. Biological therapy, originally used tofight against cancers, also plays an important role in the preventionand treatment of infections, immunodeficiency, autoimmune diseases,etc., even in transplant rejection and anti-aging.

Cancer biotherapy, by means of various bioactive agents and methods, canstimulate, activate and regulate the body's immune and anti-cancerfunctions so the body can inhibit or even eliminate the growth of tumorcells, which shows great potential in therapeutics.

The investigation and development of a therapeutic cancer vaccine is agreat challenge. Presently, satisfactory therapeutic cancer vaccineswhich can treat tumors or tumor cells effectively have still not beenobtained, and thus patients' survival time cannot be prolongedsignificantly and tumors cannot be killed reliably.

In order to establish an effective vaccine for active specificimmunotherapy, several obstacles must be overcome. Firstly, due to thepoor specificities of tumor antigens or large tumor burdens, andpatients' overall immunodeficiency caused by pre-chemotherapy as well asother factors, it is very difficult to induce the immune response ofactive specific immunotherapy which aims at tumor antigens. Secondly,because the antigenicity of tumor cells (particularly the immunogenicityof human spontaneously occurred tumors) is generally very weak, it maybe not easy for the vaccine immunization to induce an adequate immuneresponse to shrink the tumor. Thirdly, because of the heterogeneity inthe expression of the tumor antigen, most patients need to be immunizedby various antigens simultaneously. Fourthly, one of the main problemsof developing a cancer vaccine is that it is hard to build an idealanimal model which is similar to the patients' clinical situation.

Although passive specific immunotherapy, namely transferring sensitizedtumor-specific T lymphocytes, can treat the tumor of experimentalanimals effectively (confirmed in the late 60s and the early 70s), manyissues including insufficient sensitized T lymphocytes, the difficultyof rejection reaction to be induced due to the weakness of antigens ofthe spontaneous tumors, and the incompatibility of sensitized Tlymphocytes obtained from animal experiments for humans, hinder thestudy and the treatment of sensitized T lymphocytes. The biggest problemfor the application of adoptive cellular immune therapy is how to obtainsufficient sensitized tumor-specific T lymphocytes.

Active non-specific immunity can stimulate the immune system bynon-specific stimulation to enhance non-specific immune reactions; itcan improve the immune reactions to existing tumors. The majority ofactive non-specific immunotherapy applied on humans in the past is notsuccessful and most of them have not been used today. Almost all of theactive non-specific treatments for patients who had advanced cancers areunsuccessful.

Passive non-specific immunotherapy, such as LAK/IL-2 and TIL/TDAK(tumor-derived activated killing cells) therapies, mainly aims atkilling tumor cells by means of infusing their autologous or allogenicnon-specific effectors. However, the effectiveness of these therapiesare limited and need to be improved. For example, patients' insufficientautologous LAK precursor cells, along with slow amplification andlimited efficacy, all lead to the ineffectiveness of the treatment.Moreover, patients usually cannot tolerate it due to serious sideeffects brought upon by repeated applications of high-dose rIL-2. Thebiggest disadvantages of TDAK therapy (which are more obvious than thatof LAK therapy) are that it is time-consuming, laborious, and costly.The activity of TIL depends on the type, size and the extent of necrosisof the tumor, and not all tumors are infiltrated by lymphocytes. As amatter of fact, in most tumors it is difficult to obtain autologoustumor-specific TIL. The proliferative capability and anti-tumor activityof TIL will be reduced along with prolonged couture time. In addition,apoptosis can be found in some of the cells in the course of the cellculture. The TIL obtained from the tumors that produce immunosuppressivefactors may be unable to proliferate, similar to the TIL obtained frommetastatic tumors. In view of this, how to achieve these hightumor-reactive lymphocyte subsets and amplify them in vitro becomes theimportant issue of TIL/TDAK.

Therefore, it is urgent to develop a new concept and adopt a newapproach to prepare cancer vaccines and to overcome the various problemsassociated with current immune therapies mentioned above.

SUMMARY OF THE INVENTION

The present invention aims at providing a method to prepare cellpopulations with anti-tumor immune response activity and the sensitizedcell populations with anti-tumor immune response activity prepared bysaid method.

In the first aspect of the present invention, it provides a method toprepare cell populations with anti-tumor immune response activity andthe method includes:

(1) co-culturing the tumor and mononuclear cells in a three-dimensionalcell culture device, thereby obtaining the cultures which comprises cellpopulations with anti-tumor immune response activity;

(2) separating the cell populations with anti-tumor immune responseactivity from the cultures obtained from step (1).

wherein, the three-dimensional cell culture device includes:

(a) a container with liquid cell culture medium; and

(b) a three-dimensional cell culture unit in the liquid cell culturemedium, the three-dimensional cell culture unit includes empty cavitiesused for cell culture and empty cavity walls used to define the emptycavities; the empty cavity walls contain biodegradable materials towhich cells can adhere and grow; in addition, nutritional ingredientsand the products of cell metabolism can permeate the empty cavity walls;

the tumor and mononuclear cells are put together into the empty cavitiesof three-dimensional cell culture unit while they are being cultured.

In another preferable embodiment of the present invention, the tumor isselected from tumor cells, inactivated tumor cells, the cleavageproducts of tumor cells, proteins, polypeptides or other antigensobtained from tumor cells.

In another preferable embodiment of the present invention, the tumor isautologous or allogenic tumor.

In yet another preferable embodiment of the present invention, themononuclear cells are autologous or allogenic cells.

In another preferable embodiment of the present invention, the number ofthe cell populations with anti-tumor immune response activity obtainedfrom step (2) is 1×10⁵-1×10¹¹; more preferably, it is 1×10⁶-1×10¹⁰; andmost preferably, it is 1×10⁷-1×10⁹.

In another preferable embodiment of the present invention, themononuclear cells include monocytes, lymphocytes and basophilic cells.

In another preferable embodiment of the invention, the cell populationswith anti-tumor immune response activity can be made into apharmaceutical composition.

In another preferable embodiment of the present invention, thepharmaceutical composition is a vaccine.

In another preferable embodiment of the present invention, the tumorcells are obtained from the tumor tissues, malignant ascites, pleuraleffusion, or tumor cell lines of cancer patients.

In yet another preferable embodiment of the present invention, the cellpopulations with anti-tumor immune response activity include:tumor-infiltrating lymphocytes (TIL), lymphokine-activated killer cells(LAK), natural killer cells (NK), tumor-associated macrophages (TAM),activated killer monocytes (AKM), cytotoxic T lymphocytes (CTL) and/ordendritic cells (DC).

In another preferable embodiment of the present invention, theproportion of mononuclear cells and tumor cells involved in the step (2)is (5-100):1; more preferably, it is (5-50):1 and most preferably, it is(10-25):1.

In another preferable embodiment of the present invention, theco-culture time of the tumor cells and the mononuclear cells involved inthe step (2) is 3-60 days; more preferably, it is 7-28 and mostpreferably, it is 14-21 days.

In another preferable embodiment of the present invention, the materialswhich can promote the growth of mononuclear cells can be added into theculture medium involved in the step (2).

In another preferable embodiment of the present invention, thesubstances which can promote the growth of mononuclear cells arecytokines. More preferably, it is IL-2 and the concentration is 90-10000IU in general.

In yet another preferable embodiment of the present invention,antigen-presenting cells (APC) can also be added into the culture mediuminvolved in the step (2).

In another preferable embodiment of the present invention, theproportion of APC and mononuclear cells is 5:100, and more preferably,it is 1:100.

In another preferable embodiment of the present invention, theantigen-presenting cells are dendritic cells (DC).

In another preferable embodiment of the present invention, the methodalso includes: amplifying the sensitized cell populations withanti-tumor immune response activity separated from the step (2) in vitroand putting it into a container so as to form a kit.

In the second aspect, the invention provides a kind of cell populationwith anti-tumor immune response activity obtained by the method.

In the third aspect, the invention provides a kit which includes:

a container,

and the cell populations with anti-tumor immune response activitycontained in the container, which is obtained by the abovementionedmethod.

In another preferable embodiment of the present invention, the number ofthe cell populations with anti-tumor immune response activity is1×10⁵-1×10¹¹.

In another preferable embodiment of the present invention, the cellpopulations with anti-tumor immune response activity are put into theconventional infusion liquid; and in the more preferable embodiment, theconventional infusion liquid is 40 ml of 25% normal human serum albuminand 160 ml of normal saline which are put into the container.

In the fourth aspect, the invention provides the use of the cellpopulations with anti-tumor immune response activity obtained by saidmethodsin the preparation of anti-tumor drugs.

In the fifth aspect, the invention provides a method for the treatmentof tumor, it includes:

infusing the cell populations with anti-tumor immune response activityobtained by the method into the patient's body.

In another preferable embodiment of the present invention, the routes ofadministration include intravenous, pleural, abdominal cavity,intraspinal, intradermal and subcutaneous injections, injecting intotumors, etc.

In another preferable embodiment of the present invention, the cancerpatients are those who donated or those who did not donate the tumorcells for the manufacture of the cell populations with anti-tumor immuneresponse activity by the method.

In another preferable embodiment of the present invention, the cancerpatients are those who donated or those who did not donate themononuclear cells to prepare the cell populations with anti-tumor immuneresponse activity by the method.

Other aspects of the invention will be apparent to the skilled in theart in light of the technical disclosure of the invention.

FIGURE DESCRIPTION

FIG. 1 showed that the tumor cells were in close contact with themononuclear cells in the same three-dimensional cell culture device.

FIG. 2 showed the antigen collecting and the transfer process of themononuclear cell.

FIG. 3 showed the course in which the dendritic cells activate themononuclear cells.

FIG. 4 showed that a large number of proliferations and differentiationsof the mononuclear cells could be realized when it was being culturedwith the tumor cells in the same three-dimensional cell culture device.

FIG. 5 showed that two or three mature mononuclear cells move togetherto form two or three-cell units, and thus it can cooperate with eachother to implement the functions of the cell.

FIG. 6 showed the anti-tumor effects of the sensitized cell populationswith the anti-tumor immune response activity.

FIG. 7 showed the anti-tumor metastasis effects of cell populations withanti-tumor immune response activity.

FIG. 8 showed the course in which the cell populations with anti-tumorimmune response activity killed tumor cells.

FIG. 9 showed the course that cell populations with anti-tumor immuneresponse activity could lysis liver cancer cells.

FIG. 10 showed the roles played by macrophages in killing tumor cells inthe malignant pleural effusion.

FIG. 11 showed the cell populations with anti-tumor immune responseactivity attacking tumors.

FIG. 12 showed the effects of the cell populations with an anti-tumorimmune response activity sensitized by colon cancer cell lines later incontact with the colon cancer cells again.

FIG. 13 showed the sensitized cell populations with anti-tumor immuneresponse activity in the two-dimensional medium.

FIG. 14 showed the dendritic cells obtained by the method. As shown inthe figure, the morphology of the dendritic cells is similar to that ofmatured dendritic cells, with good mobility.

FIGS. 15A and 15B showed respectively the sketch map of the crosssection of the three-dimensional culture unit in the preferableembodiment of the present invention.

MODE OF CARRYING OUT THE INVENTION

After extensive and intensive studies, the inventors of the presentinvention invented a method to prepare cell populations with anti-tumorimmune response activity. The method of the present invention mainlyaims to improve the antigenicity of tumor cells, enhance thesensitization of mononuclear cells, and promote the differentiation andproliferation of the cells, while at the same time enhancing thebiological effects of sensitized immune cells. By means of said method,a large number of sensitized cell populations with high anti-tumorimmune response activity can be obtained within a short period of time.On basis of this, the present invention is completed.

As used herein, the term “cell populations with anti-tumor immuneresponse activity” is referring to the sensitized cell populations withanti-tumor immune response activity derived from mononuclear cells(which can be obtained from the peripheral blood of cancer patients andhealthy donors) after the mononuclear cells have contact with and aresensitized by tumor cells (such as tumor tissues, malignant ascites orpleural effusions or cancer cell lines). In generally, it includestumor-infiltrating lymphocytes (TIL), lymphokine-activated killer cells(LAK), natural killer cells (NK), tumor-associated macrophages (TAM),activated killer monocytes (AKM), cytotoxic T lymphocytes (CTL) anddendritic cells (DC). The cell populations with anti-tumor immuneresponse activity can also be named as a kind of “cancer vaccine”; morespecifically, it can be named as “therapeutic cancer vaccine” which canbe used to treat cancers. The “cell with anti-tumor immune responseactivity” is referred to the individual cell as the “cell populationswith anti-tumor immune response activity”.

As used herein, the term “sensitized cell with anti-tumor immuneresponse activity”, the “the cell with anti-tumor immune responseactivity” and “the immuned cell” or “the sensitized cell” areinterchangeable.

As used herein, the “mononuclear cell” is referred to as the cells withanti-tumor effects, and in general it can be obtained from theperipheral blood (from either cancer patients or healthy donors), orfrom the tumor tissues, malignant ascites, or malignant pleural effusionof cancer patients by means of discontinuous density gradientcentrifugation. The cells with anti-tumor effects mainly includelymphocytes, monocytes, basophilic cells, etc. DC can also be obtainedfrom the hematopoietic stem cells in the bone marrow.

The tumors, tumor cells and mononuclear cells adopted by the presentinvention may be autologous or allogenic.

Three-Dimensional Cell Culture Device

As used herein, “three-dimensional cell culture system”,“three-dimensional culture system”, “three-dimensional spacial culturesystem”, “three-dimensional culture device”, “three-dimensional spacialcell culture device”, “three-dimensional cell culture device” and“three-dimensional spacial culture device” are interchangeable, and allof them are referred to as the three-dimensional cell culture systemwhich is used to culture cells and provide them with a growthenvironment which is similar to an in vivo growth environment. Thisconcept can be extended to the spheriform multicellular tumor culturesystem.

The present inventors co-culture tumor and mononuclear cells in athree-dimensional culture device, the device includes:

(a) a container with liquid cell culture medium; and

(b) a three-dimensional cell culture unit in the liquid cell culturemedium, said unit includes the empty cavities which are used for cellculture and the empty cavity walls which are used to define the emptycavities; the empty cavity walls contain biodegradable materials bywhich cells can adhere and grow, in addition, some substances such asnutritional ingredients and the products of cell metabolism can permeatethe empty cavity walls.

In another preferable embodiment of the present invention, the emptycavity walls contain 80-100 wt % biodegradable materials.

In another preferable embodiment of the present invention, thecross-sectional area, length and thickness of the empty cavity are0.1-100 mm², 1-1000 mm and 0.1-10 mm, respectively. In another morepreferable embodiment of the present invention, the thickness of theempty cavity wall is 0.1-6 mm; and more preferably, it is 0.1-2 mm.

In another preferable embodiment of the present invention, the liquidcell culture medium can permeate the empty cavity walls.

In yet another preferable embodiment of the present invention, thebiodegradable materials will melt at relatively high temperatures(50-100° C.), while it is in a solid form at room temperature (25-37°C.), or in a liquid form at a low temperature (4° C.) and solidified atroom temperature (25-37° C.), respectively. The biodegradable materialsare gels which are made of biodegradable substances which are selectedfrom agar, agarose, hydrogels, collagen and matrigel, or theircombinations. Preferably, it is nonopaque, transparent andsemitransparent.

The biodegradable substances are soluble in 50-99.99% water,physiological saline solution, PBS, or cell culture medium containingextracellular matrix, growth factors, hormones and vitamins.

The shapes of the empty cavities in the invention are not limited in aparticularly way and various shapes which are suitable for the culture,contact and the interaction of tumor cells and mononuclear cells can beadopted.

Other characteristics of the three-dimensional cell culture device canalso be found in the inventors' Chinese patent application200610023537.5 or PCT patent application PCT/CN2006/000432.

Tumor Culture

The tumor cells used in the present invention can be obtained from avariety of sources such as tumor tissues, malignant ascites andmalignant pleural effusion; in addition, a certain number of tumor cellscan also be obtained by culturing the existing various tumor cell lines.

The three-dimensional cell culture device can simulate the internalenvironment to prepare a solid tumor model in vitro, and the cellbiological characteristics of the constructed tumors are similar tothose of malignant tumors in vivo. Moreover, it can secretetumor-associated antigens. The method can be used to observe thecharacteristics of tumor cells such as growth, migration, movement,invasion, metastasis, apoptosis, etc. at any time. The tumor modelprovides a unique environment for the study of cancer, and with it,various basic and clinical researches on designated tumors can becarried out.

The tumors are selected from: nasopharyngeal cancer, esophageal cancer,gastric cancer, liver cancer, breast cancer, colorectal cancer, prostatecancer, lung cancer, cervical cancer, leukemia, cancer in the oralcavity, salivary gland tumors, nasal cavity and nasal sinus cancer,laryngeal cancer, ear cancers, eye cancers, thyroid cancer, mediastinaltumors, chest wall and pleural tumors, intestinal tumors, biliary tractcancer, pancreatic and periampullary tumors, mesenteric andretroperitoneal tumors, kidney tumors, adrenal tumors, bladder tumors,prostate cancer, testicular tumor, penile cancer, endometrial cancer,ovarian cancer, malignant trophoblastic tumor, vulvar and vaginalcancers, malignant lymphoma, multiple myeloma, soft tissue tumors, bonetumors, skin and accessories tumors, malignant melanoma, nervous systemtumors and pediatric tumors.

Other characteristics of tumor culture can also be found in theinventors' PCT patent application PCT/CN2006/000432.

The Co-Culture and the Separation of Mononuclear Cells and Tumors

The present invention provides a method for preparing cell populationswith anti-tumor immune response activity, which includes: (1)co-culturing tumors and mononuclear cells in a three-dimensional cellculture device so as to obtain the cultures comprising the cellpopulations with anti-tumor immune response activity; (2) separating thecell populations with anti-tumor immune response activity from thecultures.

In the present invention, in order to obtain the sensitized cellpopulations with anti-tumor immune response activity, the proportion ofeffector cells and tumor cells is usually (5-100):1; preferably,(5-50):1; and most preferably (10-25):1. However, the proportion of thecell populations with anti-tumor immune response activity and the tumorcells may be different according to the different kinds and thedifferent tissue types of malignancy of tumors; the skilled in the artcan determine appropriate proportion by testing and evaluating thecharacteristics of the tumors.

In the present invention, the co-culture time of tumor cells andmononuclear cells is 3-60 days (more preferably, 7-28 days and mostpreferably, 14-21 days). A large number of sensitized cell populationswith anti-tumor immune response activity can be obtained in the cultureperiod. Generally, 14-21 days after culturing cells, 1×10⁵-1×10¹¹ cellpopulations with anti-tumor immune response activity can be obtaineddepending on different concentrations of initial cells.

By means of the method, the mononuclear cell stimulated by tumorantigens can excrete IL-2 or other cytokines in the course ofco-culture, so the dependency of mononuclear cell on IL-2 is relativelylow and even does not need IL-2. Exogenous IL-2, of course, can be addedinto the culture medium while the cells are being cultured, which canpromote further the proliferation of activated T lymphocytes. Forexample, the exogenous IL-2 can be added to a concentration of 90-10000IU.

A large number of cells with anti-tumor immune response activitycomprised in the cultures can be separated by the conventional methodsin the art. The method of separation adopted by the present inventionhas no special limit. For example, it can be separated by discontinuousdensity gradient centrifugation or by designing a kind of culturecondition suitable for the cells with anti-tumor immune responseactivity that allows it to grow while limiting the growth of tumor cellsso it would remove tumor cells and preserve the cells with anti-tumorimmune response activity (for example, to culture the TIL cells in atwo-dimensional culture system). Similarly, the conventional methods inthe art can also be used to amplify the cells with anti-tumor immuneresponse activity, which includes amplifying cells in the bioreactor.

To Improve the Antigenicity of Tumor Cells

In the present invention, the tumors or tumor cells are cultured withina three-dimensional cell culture device to improve the antigenicity oftumor cells. The biological characteristics and the gene expression ofsolid tumors in vivo can be mimicked by the tumor cells in vitro whichare cultured in the three-dimensional cell culture device. Therefore,the antigenicity of the tumor cells can be enhanced or improved.Compared with other existing methods, the three-dimensional cell culturedevice can provide mononuclear cells (immune cells) with an environmentsimilar to a cell-mediated immunity environment in vivo, and provide atarget of tumors similar to solid tumors in vivo, thus improving thequality and quantity of tumor antigens. These antigens are not merelyreferred to as inactivated cells, the cleavage products of cells,proteins, peptides or other antigen components. In fact, they are verysimilar to the antigens of living malignant tumors.

It is known that polypeptide antigens do not always induce the bestanti-tumor immune response, but the antigens of living tumor cellsadopted (preferably by the present invention) can induce comparativelysuperior anti-tumor immune responses. It is not necessary to know tumorantigens in the production environment of the cell populations withanti-tumor immune response activity in the present invention; the tumorantigens can be acquired by mononuclear cells themselves. In addition,although MHC-I restricted antigenic peptide can be obtained andseparated from tumor cells effectively by making use of extracts tostimulate DC, it needs a large number of tumor tissues and thecomponents of the extract are complex. Moreover, the remaining normalantigens of body will lead to the potential risk of autoimmune. Allthese disadvantages can be overcome by established tumor cell lines.

Integrated tumor cells and living tumors all have completeantigenicities, so the immune effect induced by them can target variousantigens at same time. The three-dimensional culture system not only canensure tumor cells to express fully the tumor antigens withheterogeneities, but also can provide the environment for co-stimulatoryfactors and cytokines which are necessary for the activation ofantigens. Moreover, it will be helpful to overcome possible adverseenvironments inside the body. By means of eliminating irrelevantsubstances which may inhibit immune responses and remove negativeregulation on immune cells and break immune tolerance, the quality oftumor vaccines produced will be improved as well.

The present inventors make use of autologous or allogenic tumor cells tocarry out immunoreactions in vitro, and then separates sensitizedmononuclear cells from tumor cells with the established method.Moreover, due to the tumor cells being killed gradually in the course ofthe culture, cell populations with anti-tumor immune response activity(which can be made into cell vaccine) aimed at specific tumors canfinally be obtained at the end of cell culture. The method of thepresent invention can not only fully retain the immunogenicity of tumorcells, but also can overcome “blindness”, which will be helpful toimprove the efficacy of immunotherapy.

Whether or not the functions of antigen-presenting cells (which processand present the tumor antigen initially) can be realized determines, toa large extent, the anti-tumor immune effect of cancer vaccines. Theimmune cells in the three-dimensional environment provided by thepresent invention can seek for tumor cells, capture the antigen, andactivate anti-tumor effects by themselves. The close and effectivecontact between mononuclear cells and tumor cells at the early stage ofcell culture, combined with the environment of cell-mediated immunitysimilar to the in vivo environment, as well as an effective antigenpresentation, lead to a large number of sensitized immune cells withanti-tumor immune response activity being produced.

The activation of mononuclear cells needs tumor-specific antigens ortumor-associated antigens to present antigens. Moreover, it is necessaryfor mononuclear cells to contact with tumor cells early and regularly soas to maintain its activity of cell proliferation and specific killingeffect towards the tumors and to increase the number and anti-tumoractivity of effector cells. The mononuclear cells in thethree-dimensional culture device can capture antigens continuously inthe course of the progression and development of the tumors. A long-termcell culture can ensure mononuclear cells contact with tumor cells,which can make tumor cells express tumor antigens continually and tostimulate immune cells repeatedly. This process of antigen capture willbe helpful to overcome the escape phenomenon of tumors.

To Improve the Sensitization of Immune Cells and Promote theirDifferentiation and Proliferation

The mononuclear cell can exert fully its functions by means of thethree-dimensional culture system. Only by MHC molecules presentingtumor-associated antigens for appropriate T cell subsets, can T cells beactivated in the micro-environment with co-stimulatory factors andcytokines. Therefore, it is necessary for a successful anti-tumorimmunogen to produce or trigger a local or regional activatedmicro-environment that is helpful for antigen-presenting, and T cellactivation and proliferation. However, the establishment of such amicro-environment needs the participation of “professional APCs”.Mononuclear cells, by means of cell-mediated immunity which is similarto the environment in vivo, can identify and capture antigenseffectively; for example, macrophages and dendritic cells can transmit,digest, and present antigens so as to induce the differentiation andproliferation of a large number of cells with immune activity. Therecognition of T cells on target cells is a precondition for variousimmune cells to coordinate cell-mediated immunity and to fully exertanti-tumor effects (such as mobilizing the anti-tumor effects of immuneorgans, immune cells, and immune molecules) and then a series ofeffective biological immune responses and anti-tumor effects can befully developed.

The present inventors have found that in the course of immune response,that is the generation of activated mononuclear cells that attacks andkills the tumor cells. The mononuclear cells play a cooperative role,and thus the cancer vaccine prepared by the present invention includesvarious cell populations with anti-tumor immune response activity whichis relevant with cell-mediated immunity. A variety of immune cellscooperate with each other to form a comprehensive, effective, andlasting anti-tumor immune response. The biological effects triggered bythe joint cooperation of various immune cells fully demonstrate theimportance of joint coordination between immune cells or between immunecells and other immune cells. Combined adoptive cellular immune therapyand vaccine specific immune therapy, for example, can induce sensitizedtumor-specific T lymphocytes easily, and thus improve the efficacy oftransferred effector cells. In addition, there is an immune-enhancingeffect between cytotoxic effector cells (CTL, NK, macrophages, etc.) andsecreted factors on killing tumor target cells, which is the basis of aby-stander killing effect. The secretion of cytokines and an appropriateratio of different cytokines are important to enhance immune effects.

Sufficient immune cells with specific anti-tumor effects can be obtainedat an early time of cell culture, by using the three-dimensional cellculture system. This system can also ensure various antigens withdifferent heterogeneities to induce immunity at one time, thus improvingthe quantity of the immune cells. The anti-tumor immune response inducedby the method of the present invention contains not only CTL clones witha single epitope (which may not able to induce an effective anti-tumoreffect), but also CTL clones with multiple epitopes. That means it caninduce polyclonal responses namely CD8⁺ and CD4⁺ T cell responses whichtarget various tumor antigen epitopes. The best anti-tumor effect may beobtained by means of the joint coordination of these cells. The presentinvention can make use of polyclonal or antigen-specific immune cells toidentify and kill tumor cells. Moreover, it can choose lymphocytesubsets which are more sensitive to tumor cells and amplify them invitro.

In the present invention, tumor cells and mononuclear cells areco-cultured in the three-dimensional cell culture system and the cellproliferation and differentiation of mononuclear cell can be achievedwithin 7-10 days of the culture. A large number of activated immunecells (cells with anti-tumor immune response activity) obtained at theearly time of cell culture may survive for a long time after it wastransferred into the body of the recipient. Moreover, the specificanti-tumor effect can be maintained for a period time. These immunecells will amplify rapidly and effectively when it is stimulated bytumor antigens again, and thus a specific anti-tumor immune response canbe mediated by them and finally lead to tumor rejection. Since theseimmune cells have a long life-span and will proliferate for a long timein the body, it will be helpful to obtain a long lasting immune effect.

The method provided by the present invention is a simple, convenient,ideal and natural way which accords with the normal physiologicalprocesses of the body, not needing the introduction of viruses and amodification in genes. The activated immune cells obtained by the methodare autologous or allogenic, which is helpful for the preparation andclinical application of the cancer vaccine.

To Enhance the Biological Effects of Immunocompetent Cells

The immune cells obtained by the method of the present invention showtheir immunological activity at the early days of cell culture, and itcan survive several months. The course that immune cells capture, digestand transmit tumor antigens can be observed in vitro. In addition, thecancer vaccine has various powerful immunological effects such aspreventing the growth and metastasis of tumor cells, lysis, andphagocytosing tumor cells directly.

The sensitized cell populations with anti-tumor immune response activityobtained by the present invention can carry out immune surveillance andprevent the metastasis and the proliferation of tumor cells. It willattack and kill the tumor cells once they metastasis to other places;sometimes, the cell populations with anti-tumor immune response activitywill form capsule at the edge of tumors, a morphological feature similarto benign tumors.

The examples of the present invention demonstrated that the tumor cellswere dissolved completely within several minutes after it had contactwith small sensitized lymphocytes. The effect and the course ofmacrophage killing tumor cells shows that it had contact with tumorcells first; and then, after a certain period of time, (which may be aphase that macrophages triggered and activated an immune response),tumor cells are finally phagocytosed. The present inventors alsoobserved that an autologous tumor was attacked by sensitized cellpopulations with anti-tumor immune response activity in vitro, and as aresult, the size of it reduced obviously and the tumor disappeared. Inaddition, the present inventors found that the killing effect of immunecell populations on tumor cells was associated with the reducedtumor-associated antigen. In an example of the present invention, underthe condition of the three-dimensional culture, the concentration ofalpha-fetoprotein in the liver cancer cell line ATCC-HB 8065 culturemedium without peripheral blood mononuclear cells was 1000 ng/ml, but itdecreased to 51.72 ng/ml in the culture medium when peripheral bloodmononuclear cells were added, which was reduced by 20 times. Similarly,it was 65.43 ng/ml in the culture medium of Lovo colon cancer cell linescontrol sample (without peripheral blood mononuclear cells) anddecreased to 11.33 ng/ml in the culture medium of the treated sample(with peripheral blood mononuclear cells) respectively, the latterreduced by 6 times.

The cell populations with anti-tumor immune response activity providedby the present invention includes integrated functions, namely activespecific, active non-specific, passive specific, and passivenon-specific immunities, which provides a comprehensive treatmentstrategy for the biological treatment of tumors. Combined with a humoralimmune system, better biological effects may be obtained further in theclinical treatments, including the effect on the tumor cells atquiescent cell cycle, drug-resistant cells and even on cancer stemcells.

The morphological characteristics of dendritic cells obtained by thepresent invention are similar to mature dendritic cells. Moreover, ithas a good motility. It is observed that dendritic cells depart from thetumor (obtain the antigen from the tumor), then move to adjacentmononuclear cells and have contact with them (activation). The dendriticcells with tumor-associated antigens can break immune tolerance andbring about the cytotoxic immune response of anti-tumor cell; at thesame time, the immune escape of tumor cells can be avoided. Immune cellscan amplify rapidly and effectively, mediating specific anti-tumorimmune effects and leading to tumor rejection once there is contact withtumor antigens again. The dendritic cells obtained by the method of thepresent invention can be used to prevent various solid tumors, whichwill help eventually cure human cancers.

At present, many aspects of the interaction between tumor cells andimmune cells are still unclear, but the present inventors haveestablished an ideal model to observe biological therapy of tumors invitro. By using the three-dimensional cell culture system, we canrealize a real time observation and have a dynamic understanding of themechanism of various phases of immune cells, namely the initiationphase, differentiation-inducing phase, and biological effects phase. Forexample, we can observe and understand a series of key processes such asthe acquisition, process and transfer of antigens, study the effectivemechanism for the activation of T cells (such as co-stimulatory factors)in the environment which is similar to a cell-mediated immunityenvironment in vivo, observe and study the mechanism of immune escape oftumor cell under a controllable condition, and observe biologicaleffects brought about by immune cells on tumor cells such as immuneresponse effect, immune monitoring and objective tumor regression. Themethod can also be used to separate, screen, identify and synthesizeartificially tumor antigens. Moreover, it can be applied to research theimmunogenicity of tumor antigens and improve the preparation of cancervaccine at cellular, molecular and gene levels.

The cell populations with anti-tumor immune response activity obtainedby the method of the present invention can be used for active specificimmunotherapy. The present inventors, based on the theoretical basis ofactive specific immunity, provide a new method to study tumor vaccinesto improve its specificity, safety and efficiency: 1) culture immunecells in the three-dimensional environment so as to make it to seek fortumor antigen spontaneously, 2) prepare tumor vaccines under acontrollable condition in vitro (modification of cytokines, immuneadjuvant, immune regulator), which can break the body's immune toleranceon tumors, relieve immunosuppression, and avoid or overcome T cellanergy, 3) through the cooperative effect between various immune cells,the comprehensive anti-tumor effect of cell-mediated immunity can beenhanced, which includes that tumor cells are dissolved directly by CD8⁺CTL; the direct killing effect of tumor cells such as tumor cells bedigested by various enzymes. Moreover, CD4⁺ T cell can release cytokinesto kill tumor cells or inhibit their growth indirectly or directly.

The method invented by the present inventors overcome various currentrestrictions on the clinical application of DC: 1) by means of themethod provided by the present inventors, macrophages and lymphoid toxiccells can kill and lysis tumor cells so as to provide various suitabletumor antigens for DC; 2) the MHC restriction can be avoided by adoptingautologous DC; 3) in the production environment of a cancer vaccine, thepresent inventors do not always need to know tumor antigen, in fact,immune cells can capture tumor antigens independently; 4) it is knownthat the best anti-tumor immune response can not always be induced bypolypeptide antigens alone, however, the antigens of the living tumorcells adopted by the present inventors can induce an ideal anti-tumorimmune response; 5) the method provided by the present invention notonly targets single epitope CTL clones, which may not able to induce aneffective anti-tumor effect, but also can obtain CTL clones withmultiple epitopes; the best anti-tumor effect can be realized by meansof the combined effect of these cells. In addition, although MHC-Irestricted antigen peptide separated and obtained from tumor cells hascertain effects, the method that makes use of extract to stimulate DCneeds a lot of tumor tissues. Moreover, the antigen of extract hascomplex components and there is a potential risk that the normalantigens of body will lead to autoimmunity. All these disadvantages canbe overcome by established tumor cell lines.

The method adopted by the present inventors is that making sure of theantigens of tumor cell to activate immune cells, which differs from themethod that involves activating TIL with IL-2 alone. This method canensure tumor cells contact with immune cells at the early stage of thecell culture, and combined with the mediated role played by other Tcell-associated molecules, the effective tumor tumor-specific antigen ortumor-associated antigen can be provided. In the course of long-termcell culture, immune cells closely contact with the tumor so as tomaintain its activities, namely cell proliferation and a specifickilling effect on autologous tumors, through a specific activationmechanism. Since the T cells activated by this mechanism can excreteIL-2 and other cytokines when it is stimulated by antigens, it shows alower dependence on IL-2 and the amount of IL-2 to be added is small.The T cell can survive for a long time and maintain its specificanti-tumor immune response after it was infused into the body of therecipient. Moreover, it can amplify rapidly and effectively, mediatespecific anti-tumor immune response, and reject the tumor once it isstimulated by tumor antigen again.

Theoretically, making use of sensitized tumor-specific T lymphocytes totreat tumor has great advantages, which has been proved by animalexperiments; however, it is still very difficult to apply it in theclinical treatment due to several technical reasons. The method providedby the present inventors overcame existing difficulties, which hasadvantages as follows: 1) high frequency of effector cell precursors andtimesaving in vitro culture and amplification with low cost, 2) theantigenicity of tumors in the course of evolution changes frequently(such as antigen modulation), which will lead to some tumor cells toescape from the attack of T lymphocytes and grow continuously, and thusbecome the seeds of the recurrence and metastasis of tumor; thesensitized cells in the system provided by the present inventors canreact continuously with the tumor cells, which can produce correspondingimmune responses for changed tumor cells to avoid tumor escape, 3)immune response can be induced by autologous immune cells to overcomethe MHC restriction on the anti-tumor effect of T lymphocytes, which maynot be realized by allogenic T lymphocytes.

The method provided by the present inventors overcame many restrictionsor shortcomings of LAK/IL-2 therapy such as the small number and the lowamplification of LAK precursors and the need of taking a large number ofleukocytes from the body, which will lead to infection easily and weakenthe body's immune system. In addition, the immunosuppression broughtabout by the growth of the tumor lead to poor quality of LAK cellsinduced by patient's leukocytes—all these defects above can be overcomeby allogenic mononuclear cells. Since the method belongs to non-specificimmune response, it has a limited killing effect. Applying high-doseIL-2 repeatedly will frequently cause serious side effects which willmake patients unable to tolerate treatment; the method used by thepresent inventors, however, includes various mechanisms. A large numberof mononuclear cells produced by the method can excrete endogenous IL-2,which can improve the in vitro culture and the inducement of LAK cells;it can improve efficacy and reduce treatment cost, and thus make thisLAK/IL-2 therapy more accepted by the patients.

The biggest problems of TIL/TDAK therapy are as follows: 1) cell cultureis very time-consuming, laborious, expensive and easily contaminated,which is more obvious than that of LAK cells, 2) the activity of TILrests with the tumor's type, size and the degree of necrosis; not alltumors can be infiltrated by lymphocytes, 3) the proliferative andanti-tumor activities of TIL will reduce with prolonged culture time;apoptosis can be found in some cells in the course of cell culture, 4)it is difficult to obtain self-tumor-specific TIL from most tumors;moreover, the TIL obtained from metastatic tumors or tumors which cansecrete immunosuppressive factors may not be amplified in vitro; thetumor infiltrating lymphocyte has diversity, which is determined by theimmunity of tumor cells. The anti-tumor effect of TIL will be diminishedby the abnormal differentiation and the shortage of cytokine or tumorfree components which are necessary for TIL to exert killing effects.The biggest challenge faced by TIL cell therapy is how to obtain a largenumber of early effectors to carry out adoptive cell-mediatedimmunotherapy, which has been overcome by the present inventors. Bymeans of the method provided by the present inventors, a large number ofearly stage anti-tumor immune active cells can be obtained within 2-3weeks, which is far less than that of a TIL cell culture (in general,the culture time is 45-60 days).

The three-dimensional cell culture system can also be used to preparevarious cancer vaccines such as embryonic antigen vaccines, virusvaccines, cancer gene products, synthetic peptide vaccines,anti-idiotypic antibody vaccines and genetically engineered vaccines. Atthe same time, it is helpful for the study of immune adjuvants such asnon-specific immune adjuvants, the gene modification of tumor cells andpeptide-assisted T helper cell immune response; moreover, it can offerco-stimulatory signals and cytokines for T lymphocytes and present theantigens which are coded by recombinant plasmid and the bacterial orvirus.

In addition, the present invention also provides a kit which includes acontainer; the container comprises the cell populations with anti-tumorimmune response activity obtained by the method. The number of the cellpopulations in the preferred embodiment of the present invention is1×10⁵-1×10¹¹. At last, various existing methods in the art can be usedto transfer the cell populations into an infusion solution such as 40 ml25% normal serum albumin and 160 ml normal saline. More preferably, thecontainer also includes an operation manual.

The present invention also provides a use of the cell populations withanti-tumor immune response activity obtained by the method; it can beused to prepare cell therapy drugs for tumor.

The present invention also provides the methods for the treatment oftumors, it includes infusing the cell populations with anti-tumor immuneresponse activity obtained by the method into cancer patient's body,wherein, the cancer patient may be or may not be the patient who donatestumor cells for the method of the present invention. The cancer patientmay be or may not be the patient who donates mononuclear cells for themethod of the present invention.

The dosage and administration route of the cell population withanti-tumor immune response activity should be chosen according to thepatient's individual characteristics and the degree of disease; forexample, infusing the 1×10⁵-1×10¹¹ anti-tumor immune cells in topatient's body for about 7 days, every other 1-3 weeks. Of course,specific dosage should be decided by means of other factors such asadministration route, health status, etc., and all these skills are notbeyond the scope managed by skilled medical doctors.

The method of the present invention can be used alone; it can also becombined with other tumor therapies. The tumor therapies include, forexample, surgery, chemotherapy, radiation therapy and other biologicaltherapies.

The method provided by the present invention may play an important rolein the prevention and the treatment of other diseases such as infection,immunodeficiency, autoimmune disease, etc., and even in the areas oftransplant rejection and anti-aging therapy.

The present invention will be further illustrated with the followingexamples. It should be understood that, these examples are exemplaryonly and are not intended to limit the scope of the present invention.The experimental methods in the following examples not indicating thespecific experimental conditions are typically carried out under theconventional conditions, for example, those in Sambrook, et al.Molecular cloning: A laboratory manual (New York: Cold Spring HarborLaboratory Press, 1989), or following the manufacture's instructions.Unless otherwise indicated, all the percents and parts are calculated byweight.

General Materials and Methods

1. Construction of Three-Dimensional Culture System

To construct a three-dimensional culture system, the three-dimensionalculture system includes: a container with liquid cell culture medium andthree-dimensional cell culture units in the liquid cell culture medium,in which the three-dimensional cell culture unit has an empty cavity andempty cavity walls which are used to define the empty cavity, the emptycavity walls contain biodegradable materials on which cells can adhereand grow, and moreover, nutrients components and cell metabolites canpass through the empty cavity wall.

(1) In a preferable method, the empty cavity walls of thethree-dimensional cell culture units are made of 1% agarose, and thecross-section of it is circular or approximately circular. A circularmold with a jacket pipe (external diameter: about 6 mm, length: about200 mm) is used to prepare the three-dimensional cell culture units. 5round wires (diameter: 0.3 mm, length: 300 mm) are arranged evenly inthe internal cavity of the mold, which is filled with 1% agarose andafter the agarose solidified, once taking out the wires and the mold, athree-dimensional cell culture unit (external diameter: about 5.8 mm)with 5 symmetrical internal cavities can be obtained. The sketch map ofthe cross-section is shown in FIG. 15A, wherein, a) denotes the emptycavity wall, b) denotes empty cavities. The three-dimensional cellculture units will be put into the liquid culture medium which issuitable for the growth of mononuclear cells and tumor cells.

(2) In another preferable method, the empty cavity walls of thethree-dimensional cell culture units are made of 1.2% agarose; thecross-section of it is quadrate or approximately quadrate. Beforepreparing the three-dimensional cell culture unit, a cuboid mold with ametallic jacket pipe (length: 10 mm, width: 6.5 mm, height: 3 mm, thethickness of wall: 0.5 mm) is designed first; 3 quadrate wires (length:20 mm, width: 0.9 mm, height: 0.9 mm) are arranged in the jacket pipe.The two ends of the jacket pipe are sealed with movable covers which arematched with the jacket pipe. After filling the mold with 1.2% agaroseand taking out the wires and the mold after the agarose solidified, thethree-dimensional cell culture unit (length: 10 mm, width: 5.5 mm,height: 2 mm) with 3 symmetrical internal cavities (0.9×0.9 mm) can beobtained. The sketch map of the cross-section is shown in FIG. 15B,wherein, 1) denotes the empty cavity wall and 2) denotes empty cavities.

The three-dimensional cell culture system can provide an environmentwhich is similar to the cell-mediated immune environment in the body forthe growth and the movement of tumor cells and mononuclear cells, whichprovides a perfect space and environment for them to contact or interactwith each other.

2. The Separation of Mononuclear Cells (Non-Continuous Density GradientCentrifugation)

The peripheral blood specimens (3 ml) anticoagulated by EDTA wascollected by means of aseptic method, and cells were separated withlymphocyte separation medium (Ficoll-Hypaque, Sigma Inc) andnon-continuous density gradient centrifugation. The concrete steps wereas follows: adding 3 ml of 100% lymphocyte separation medium to the testtube, then adding 3 ml of a blood sample carefully on top of thesolution, (along the wall of the test tube, these two parts of thesolution should not be mixed), centrifugating it 25 min at 1500 r/min,collecting cells from the cell layer at the interface of the lymphocyteseparation medium, and washing cells in turn with sterile buffer andRPMI1640 one time to obtain mononuclear cells. The actual number ofspecimens can be determined according to the number of cells which willbe used.

The results from the test showed that the extracted mononuclear cellsmainly include: mononuclear cells, lymphocytes, and basophilic cells.

Lymphocyte separation medium (density: 1.068) and non-continuous densitygradient centrifugation can be used to separate DC.

3. The Extraction of Tumor Cell (Specimen)

A. From Malignant Pleural Effusion or Ascites

Tumor cells, conventionally, can be extracted from malignant pleuraleffusion or ascites, wherein, pleural effusion is suitable for theextraction of specimen of lung cancer and other secondary tumors,ascites is suitable for the extraction of specimens of digestive tracttumors, ovarian cancers, etc.

The patients, who, (confirmed by pathological cytology) suffered frommalignant pleural effusion (such as lung cancer, breast cancer) ormalignant ascites (such as colon cancer, liver cancer) can be enrolledto extract the specimens of tumor cells.

Taking pleural effusion or ascites 500-1000 ml from patients by means ofaseptic method and pleuracentesis, the separation and preparation test,after adding heparin 10 U/mL into it, should be carried out immediately.One should centrifugate malignant pleural effusion or ascites withheparin 10 min at 1500 r/min (400 g). Then wash deposited cells with asterile buffer twice to hemorrhagic pleural effusion or ascites, theconcrete steps are described as follows: adding erythrocyte lysingsolution (volume ratio: 1:10) into it and mixing it evenly; storing itat 4 C, 8 min; centrifugating it 5 min at 1500 r/min; washing depositedcells in turn with sterile buffer and RPMI1640 culture medium one time;suspending cells (1×10⁶/mL) in the RPMI1640 culture medium (containing10% FBS) and putting it into the three-dimensional cell culture devicefor cell culture; the culture conditions: saturated humidity, 37 C, 5%CO₂.

B. Making Use of Tumor Cell Lines to Culture Tumor or Tumor Cell

By putting the selected cancer cell lines into the three-dimensionalcell culture device described in “1” and adding appropriate culturemedium into it to culture cells 3-7 days, then a certain number of tumorcells or micro-tumors can be obtained for use.

4. The Co-Culture of Tumor Cell and Mononuclear Cell in theThree-Dimensional Culture Device

The tumor cells obtained from “3” are co-cultures with the mononuclearcells obtained from “2” in the three-dimensional culture device whichwas constructed by “1” mentioned above. The concentration of tumor celland mononuclear cell is 5-50:1 (such as 25:1, namely, taking one tumorcell to activate 25 mononuclear cells so as to form a cancer vaccine), apreferable culture time is 14-21 days.

5. The Separation and the Culture of Sensitized Cell Populations withAnti-Tumor Immune Response

Taking out the mixture of tumor cells and cell populations withanti-tumor immune response from the three-dimensional culture device,centrifugating it for 10 min at 1000 r/min, washing deposited cells witha sterile buffer twice and suspending it in buffer 3 ml; separatingcells with lymphocyte separation medium and non-continuous densitygradient centrifugation. The concrete steps were as follows: adding inturn of 100% and 75% lymphocyte separation medium 3 ml into the bottomand the middle-layer of test tube respectively; adding the cellsuspension 3 ml carefully into test tube along the wall of test tube,which should not be mixed; centrifugating it 25 min at 2000 r/min (700g) and collecting cell populations with anti-tumor immune response from100% liquid interface. Centrifugating cell populations 5 min at 1000r/min and washing deposited cells in turn with sterile buffer andRPMI1640 one time so as to obtain cell populations with anti-tumorimmune response; transferring cell populations on a 6-well plate andculturing it for 48 h in an incubator (5% CO₂, 37 C); transferring theminto a cell culture bottle and adding AIM-V serum-free culture medium(Gibco) with IL-2 1000 U/ml into the culture bottle.

6. The Culture and the Cell Proliferation of Cell Populations withAnti-Tumor Immune Response

The cell populations with anti-tumor immune response can be proliferatedfurther by means of suspending cells (1.0×10⁶/ml in the AIM-V serum-freeculture medium with IL-2 1000 U/ml, which can be regarded as an optionalway for cell proliferation. The cell populations can be cultured in thebig culture bottles (175 cm²) or culture bags (750 cm²) so as to meetthe need of large-scale culture for clinical treatment. Generally,subculture should be carried out once a week, which includes thereplacements of fresh medium and rIL-2, adjusting cell concentration to5.0×10⁵/ml, and culturing them continuously in a new container. Inaddition, the cell populations with anti-tumor immune response can alsobe proliferated in a bioreactor.

Example 1 The Close Contact Between the Tumor Cells and MononuclearCells

Co-culturing liver cancer cell lines SMMC 7721 (Purchased from Cell Bankof Chinese Academy of Sciences) and peripheral blood mononuclear cellsobtained from healthy donors in the three-dimensional culture unit (1)constructed by the method “1”; the composition of the culture mediumincludes: RPMI 1640 (Sigma Inc) 1000 ml, 100× penicillin+streptomycin(Gibco 15140-122) 10 ml and fetal bovine serum 100 ml. Culturecondition: 37 C, 5% CO₂. Similar culture condition is used for coloncancer cell lines Lovo (Purchased from ATCC) and peripheral bloodmononuclear cells obtained from healthy donors; the culture mediumincludes: 1000 ml Ham's F12K (Gibco 21700-026) culture medium withL-glutamine 2 mM, 100× penicillin+streptomycin (Gibco 15140-122) 10 ml,10× glutamine 10 ml, fetal bovine serum 100 ml. Adjusting theconcentration of mononuclear cells and tumor cells to 10:1, thenobserving the interrelationship between them by means of IX71 invertedmicroscope (Olympus).

The results showed that the mononuclear cells contacted closely withliver cancer cells and colon cancer cells within 24-48 h while they werecultured in the three-dimensional culture system. It is shown in theFIGS. 1A-D (liver cancer cells and mononuclear cells; 40×) and FIG. 1E-H (colon cancer cells and mononuclear cells; E, F: 40×; G, H: 20×); inthe figures, I denotes cancer cell, II denotes mononuclear cell.

Example 2 The Collection and the Transfer of Antigen by Lymphocytes

Adding colon cancer cell lines Lovo and peripheral blood mononuclearcells obtained from healthy donors into the three-dimensional cultureunit (1) constructed by the method “1”, then culturing it in the culturemedium; the composition of the culture medium is same with that ofexample 1, adjusting the concentration of mononuclear cell and tumorcell to 10:1, then observing the interrelationship between them.

FIG. 2 A-H (40×) shows the phenomenon that at least two lymphocytescooperated with the collection and the transfer of surface antigens ofcolon cancer cells at the 8^(th) day of cell culture; wherein, the onelymphocyte captured antigens at the surface of tumor cell and thecaptured antigen was transferred by another one. FIGS. 2A-H showed thedynamic course of the collection and the transfer of antigens, whichlasted 86 min; in the figures, I denotes colon cancer cells, II denoteslymphocytes.

Example 3 The Phenomenon that Dendritic Cell Activates Lymphocyte

Obtaining tumor cells and mononuclear cells from the malignant pleuraleffusion of a patient who suffered from lung cancer by means of themethod; mixing the obtained cells with autologous peripheral bloodmononuclear cells 3 ml and putting them into the empty cavity ofthree-dimensional culture unit (2) constructed by the method “1”; thecomposition of the culture medium includes: RPMI 1640 (Sigma Inc.) 1000ml, 100× penicillin+streptomycin (Gibco 15140-122) 10 ml and fetalbovine serum 100 ml. Culture conditions: 37° C., 5% CO₂.

The results showed that dendritic cells, at the 9^(th) day of culture,departed from tumors (extracted antigen from tumor) and moved tosurrounding lymphocytes so as to contact with (activate) them. FIGS.3A-H showed the dynamic course that dendritic cell activateslymphocytes, which lasted 94 min; in the figures, I denotes dendriticcells, II denotes lymphocytes.

Example 4 The Proliferation and the Differentiation of Mononuclear Cell

Co-culturing colon cancer cell lines Lovo and liver cancer cell linesSMMC 7721 respectively with peripheral blood mononuclear cells obtainedfrom healthy donors into the three-dimensional culture unit (1)constructed by the method “1”; the methods of culture are same as theformer method. The proliferation and the differentiation of mononuclearcells can be observed at 7-10^(th) day of cell culture.

FIGS. 4A-B (A, 40×; B, 20×) shows the conditions of growth of coloncancer cells and mononuclear cells observed at the 7^(th)-10^(th) day ofcell culture in the three-dimensional culture device, from which a largeamount of proliferation of mononuclear cells can be found. In thefigure, I denotes colon cancer cells, II denotes mononuclear cells.

FIGS. 4C-D (40×) showed the conditions of growth of liver cancer celland mononuclear cell observed at 14^(th) day of cell culture in thethree-dimensional culture device, the differentiation of mononuclearcells can be observed.

Example 5 The Congregation and the Cooperation of Mononuclear Cell

Obtaining tumor cells and mononuclear cells from the malignant ascitesof patient who suffered from colon cancer by means of the mentionedmethod; mixing the obtained cells with autologous peripheral bloodmononuclear cells 3 ml and putting them into the empty cavity ofthree-dimensional culture unit (1) constructed by the method “1”; thenculturing the three-dimensional culture unit in the culture medium, thecomposition of the culture medium includes: 1000 ml Ham's F12K (Gibco21700-026) culture medium with L-glutamine 2 mM, 100×penicillin+streptomycin (Gibco 15140-122) 10 ml, 100×glutamine 10 ml andfetal bovine serum 100 ml.

Co-culturing breast cancer cell lines (ATCC HTB-22) and peripheral bloodmononuclear cells obtained from healthy donors in the three-dimensionalculture unit (1) constructed by the method “1”; then culturing thethree-dimensional culture unit in the culture medium, the composition ofthe culture medium includes: Minimum essential medium (Eagle) with 2 mMof glutamine and Earle's BSS (Gibco 11700-077), sodium bicarbonate 1.5g/L, non-essential amino acids 0.1 mM, sodium pyruvate 1 mM, bovineinsulin 0.01 mg/ml, 100× penicillin+streptomycin (Gibco 15140-122) 10 mland fetal bovine serum 100 ml. Culture conditions: 37 C, 5% CO₂.Adjusting the concentration ratio of mononuclear cell and tumor cell to10:1.

Similarly, culturing colon cancer cell lines Lovo and peripheral bloodmononuclear cells obtained from healthy persons in the three-dimensionalculture unit (1) constructed by the method “1”.

The phenomenon that two or three mononuclear cells got together can beobserved at 10-14^(th) day of cell culture, see FIG. 5A (ascitesspecimen, 40×); FIG. 5B (the specimens of mononuclear cell and breastcancer cells, 40×); FIGS. 5C-D (the specimens of mononuclear cells andcolon cancer cells, 40×).

From the results above, we can see that two or three mature mononuclearcells stay together and form a combined two-cells unit or a combinedthree-cells unit of mononuclear cells to cooperate with each to executethe cellular functions.

Example 6 The Anti-Tumor Effect of Sensitized Cell Populations withAnti-Tumor Immune Response Activity

Co-culturing liver cancer cell lines (SMMC 7721) and peripheral bloodmononuclear cells obtained from healthy donors in the three-dimensionalculture unit (1) constructed by the method “1”; the methods of cultureare the same as the former method. At the 7^(th)-14^(th) day of cellculture, a great deal of cells with anti-tumor effect adhered to andcovered the surfaces or the edges of tumors to inhibit the growth andthe metastasis of tumors; the sensitized cells could also form a “fence”to prevent the metastasis of the tumor cells, see FIGS. 6A-B (20×), ormonitor the transfer of tumor cells at the edge of tumor, see FIGS. 6C-E(40×). Once tumor cells starts metastasis, the sensitized cells couldmove toward the tumor cells and block them from departing from thetumor, see FIG. 6E. In the figures, I denotes liver cancer cell, IIdenotes cell populations with anti-tumor immune response activity.

Obtaining tumor cells and mononuclear cells from the malignant pleuraleffusion of patients who suffered from lung cancer by means of thementioned method; mixing the obtained cells with autologous peripheralblood mononuclear cells 3 ml, adding them into the empty cavity ofthree-dimensional culture unit (1) constructed by the method “1”, thenculturing the three-dimensional culture unit in the culture medium andobserving the interrelationship between sensitized cells and tumorcells. On the 14^(th) day of cell culture, it can be observed that thecells with anti-tumor immune response activity were stationed at theedges of tumors and monitored the metastasis of tumor cells, see FIG. 6F(40×). In the figure, I denotes tumor cells, II denotes cells withanti-tumor immune response activity.

Co-culturing colon cancer cell lines Lovo and peripheral bloodmononuclear cells obtained from healthy donors in the three-dimensionalculture unit (1) constructed by the method “1”, then culturing thethree-dimensional culture unit in the culture medium and observing theinterrelationship between sensitized cells and colon cancer cells; themethods of culture are the same as former methods. At the 14^(th) day ofcell culture, it can be observed that metastasis tumor cells can also bekilled by the cell populations with anti-tumor immune response activity,see FIGS. 6G-H (40×). Sometimes, the cell populations with anti-tumorimmune response activity could form “capsule” at the edge of tumors,which led to malignant tumors to show the morphological characteristicssimilar to benign tumors, see FIG. 6G. In the figure, I denotes coloncancer cell, II denotes cells with anti-tumor immune response activity,III denotes capsule.

Example 7 The Anti-Tumor Metastasis Effect by Cell Populations withAnti-Tumor Immune Response Activity

Co-culturing colon cancer cell lines Lovo and peripheral bloodmononuclear cells obtained from healthy donors in the empty cavities ofthree-dimensional culture unit (1) constructed by the method “1”, thenculturing the three-dimensional culture unit in the culture medium andobserving the interrelationship between sensitized cells and coloncancer cell; the methods of culture are same with former method.

On the 9^(th) day of cell culture, we observed the phenomenon in whichsensitized cells block the gap at which tumor cell metastasis occurred.It can be showed in the FIGS. 7A-H (20×) in which sensitized cells movedto the place at which tumor cell metastasis occurred, then appeared torelease a small lymphocyte and some substances yet to be identified (maybe a kind of substance for the attachment) through a “pipeline” which isformed by some unknown substances around the tumor; then the smalllymphocyte moved to and stationed at the edge of the tumor to preventtumor cells from metastasis again. At the same time, three sensitizedcells had also moved to and stationed at the gap at which tumor cellsmetastasis occurred. FIGS. 7A-H showed the dynamic course of anti-tumoreffects which lasted 74 min; in the figures, I denotes colon cancercell, II denotes sensitized cell, III denotes small lymphocyte, IVdenotes viscous substance, V denotes pipeline.

Example 8 The Course that Cell Populations with Anti-Tumor ImmuneResponse Activity Kill Tumor Cells

Co-culturing breast cancer cell lines (ATCC HTB-22) and peripheral bloodmononuclear cells obtained from healthy donors in the three-dimensionalculture unit (1) constructed by the method “1”. The methods of cultureare the same as the former method.

FIGS. 8A-H (40×) showed that the course in which two breast cancer cellswere killed and phagocytized by a number of sensitized cells at 10^(th)day of culture can be described as follows: firstly, sensitized cellsperforated on the tumor cells, then the cytoplasm of tumor cells flowedout along the perforation; in succession, other sensitized immune cellscontacted with the breast cancer cells to attack them, (for example, itpeeled off part cell membrane of the tumor cells) and at last, the tumorcells were phagocytized by sensitized or activated macrophages. FIGS.8A-H showed the course that cell populations with anti-tumor immuneresponse activity killed tumor cells, which lasted 130 min. In thefigures, I denotes breast cancer cells, II denotes sensitized cells, IIIdenotes macrophages.

Example 9 The Course that Cell Populations with Anti-Tumor ImmuneResponse Activity Dissolve a Liver Cancer Cell

Co-culturing liver cancer cell lines SMMC 7721 and peripheral bloodmononuclear cells obtained from healthy donors in the three-dimensionalculture unit (1) constructed by the method “1”, then observing theinterrelationship between sensitized cells and liver cancer cell. Themethods of culture are same with former method.

FIGS. 9A-H (40×) showed the course in which several sensitized cellswith anti-tumor immune response activity lysis a liver cancer cell at4^(th) day of culture. It can be observed that the liver cancer cell wasdissolved within several minutes when it contacted with sensitizedcells. FIGS. 9A-H showed the dynamic course that cell populations withanti-tumor immune response activity lysis a liver cancer cell, whichlasted 10 min. In the figures, I denotes liver cancer cells, II denotessensitized cells.

Replacing SMMC 7721 with ATCC-HB 8065 cell line and culturing it withperipheral blood mononuclear cells according to same method mentionedabove. After 10 days of culture, the concentration of AFP in the culturemedium of processed specimen reduces to 51.72 ng/ml, but it is 1000ng/ml in the control specimen (the culture medium of liver cancer celllines ATCC-HB 8065 without peripheral blood mononuclear cells; otherculture conditions and culture time are the same as former method).Conventional immunoassay was used to determine the concentration of AFP.

Example 10 The Tumor-Killing Effect of Macrophage in the MalignantPleural Effusion

Obtaining tumor cells and mononuclear cells from the malignant pleuraleffusion of patient who suffered from lung cancer by means of themethod, then mixing it with autologous peripheral blood mononuclearcells 3 ml; putting the mixture into the empty cavities ofthree-dimensional culture unit (1) constructed by the method “1” andculturing it in the culture medium.

FIGS. 10A-H (40×) showed the course that macrophages kill tumor cells:firstly, macrophages have contact with tumor cells, 3 hours and 2minutes later (presumably by triggering immune response, which startedto activate immune cells), the tumor cells were killed finally by themacrophages. FIGS. 10A-H showed the dynamic course that macrophages killtumor cells. In the figures, I denotes tumor cell, II denotesmacrophage.

Example 11 The Tumor-Killing Effect of Cell Populations with Anti-TumorImmune Response Activity

Obtaining tumor cells and mononuclear cells from the malignant pleuraleffusion of patient who suffered from lung cancer by means of themethod, then mixing it with autologous peripheral blood mononuclearcells 3 ml; putting the mixture into the empty cavities ofthree-dimensional culture unit (1) constructed by the method “1” andculturing it in the culture medium.

The results showed the course in which the tumor cells in the malignantpleural effusion were attacked by the sensitized cell populations withanti-tumor immune response activity; the tumor size was reducedobviously and finally disappeared; see FIG. 11 (40). FIGS. 11A-H showedthe dynamic course of the tumor-killing effect of cell populations withanti-tumor immune response activity, which lasted 3 days. In thefigures, I denotes tumor cells, II denotes cell populations withanti-tumor immune response activity.

Example 12 The Biological Effects of Sensitized Cells Activated by ColonCancer Cell when it Contacts with Colon Cancer Cell Again

Adding colon cancer cell lines Lovo and peripheral blood mononuclearcells obtained from healthy persons into the three-dimensional cultureunit (1) constructed by the method “1”; then culturing it in the culturemedium; the composition of the culture medium is same as the formermethod; at 14^(th) day of cell culture, making use of the method toseparate mononuclear cells with anti-tumor immune response activity fromthe culture, one then observes the biological effects of sensitizedcells activated by colon cancer cell when it has contact with the coloncancer cell again in the three-dimensional culture unit.

From FIG. 12, we can see that two mature sensitized cells got togetherand formed a combined unit of sensitized cells (FIG. 12A, 40×); a closecontact between the colon cancer cells and the mononuclear cells can beestablished within 2 hours of culture in the three-dimensional culturedevice (FIG. 12B, 40×); 10 days after cell culture, the sensitized cellswith anti-tumor effect adhered and covered on the surfaces or at theedges of tumor cells so as to prevent the growth and the transfer oftumor cells (FIG. 12C, 40×); moreover, it can also form capsule at theedges of tumor, which led to malignant tumors showing the morphologicalcharacteristics similar to that of benign tumor (FIG. 12D, 40×); 20 dayslater, a large number of colon cancer cells Lovo died (FIGS. 12E-F,20×). In the figures, I denotes tumor cells, II denotes sensitizedcells, III denotes capsule.

At the same time, the concentration of carcinoembryonic antigen in theculture medium of processed specimen was 11.33 ng/ml, but it was 65.43ng/ml in the control specimen (the culture medium of colon cancer celllines Lovo without peripheral blood mononuclear cells; other cultureconditions and culture time are the same as the former method).Conventional immunoassay was used to determine the concentration ofcarcinoembryonic antigens.

Example 13 The Culture of Sensitized Cell Populations with Anti-TumorImmune Response Activity in the Two-Dimensional Culture System

Separating sensitized cell populations with anti-tumor immune responseactivity cultured in the three-dimensional culture system from tumorcells and culturing them in the two-dimensional culture system, thenobserve the growth of cells which is showed in the FIGS. 13A-B (A, 20×;B, 40×).

From FIG. 13, we can see that a large number of immune cells can befound in the two-dimensional culture system; in addition, there were notumor cells in the culture system. These immune cells can be collectedand amplified further to appropriate concentration to meet the needs ofclinical application.

Example 14 The Preparation of Dendritic Cell

Obtaining tumor cells and mononuclear cells from the malignant pleuraleffusion of patient who suffered from lung cancer by means of themethod, then mixing it with autologous peripheral blood mononuclearcells 3 ml; putting the mixture into the empty cavities ofthree-dimensional culture unit (2) constructed by the method “1” andculturing it in the culture medium.

From FIGS. 14A-H, we can see that the morphological characteristics ofdendritic cell were similar to mature DC after 21 days of culture.Moreover, it had a good motility. In the figures, III denotes dendriticcells. These cells can be collected and amplified further to appropriateconcentration so as to meet the needs of clinical treatment.

All the documents cited herein are incorporated into the invention asreference, as if each of them is individually incorporated. Further, itwould be appreciated that, in light of the above described teaching ofthe invention, the skilled in the art could make various changes ormodifications to the invention, and these equivalents would still bewithin the scope of the invention defined by the appended claims of theapplication.

The invention claimed is:
 1. A method for preparing cell populationswith anti-tumor immune response activity, wherein the method includes:(1) co-culturing tumor cells and mononuclear cells for 7-28 days in athree-dimensional cell culture device, thereby obtaining a culture whichcomprises cell populations with anti-tumor immune response activity; (2)separating the cell population with anti-tumor immune response activityfrom the culture which is obtained by step (1); wherein, thethree-dimensional cell culture device includes: (a) a container withliquid cell culture medium; and (b) a three-dimensional cell cultureunit in the liquid cell culture medium, the three-dimensional cellculture unit is formed of biodegradable material which melts at atemperature of about 50° C. to about 100° C. and are solid at atemperature of 25° C. to about 37° C.; comprises one or more emptycavities used for cell culture, wherein the empty cavity walls definethe empty-cavities; the empty cavity walls are comprised of abiodegradable material to which cells can adhere and grow and the cavitywalls are permeable to the nutritional substances and the products ofcell metabolism; wherein the biodegradable material of the cavity wallsof the three dimensional culture device is nonopaque and is at least oneselected from the group consisting of agar, agarose, hydrogels,collagen, and matrigel; and wherein the tumor cells and mononuclearcells are put together in the one or more empty cavities of thethree-dimensional cell culture unit within the cell culture mediumthereby forming a co-culture of tumor cells and mononuclear cellsresulting in a cell population with anti-tumor immune response activity.2. The method of claim 1, wherein the cell population with anti-tumorimmune response activity comprises one or more of: tumor-infiltratinglymphocytes, lymphokine-activated killer cells, natural killer cells,tumor-associated macrophages, activated killer monocytes, cytotoxic Tlymphocytes and/or dendritic cells.
 3. The method of claim 1, whereinthe co-culture of tumor cells and mononuclear cells has a ratio ofmononuclear cells to tumor cells which is in the range of 5:1 to 100:1.4. The method of claim 1, further comprising adding a substance that canpromote the growth of mononuclear cells into the liquid cell culturemedium in the three-dimensional cell culture device in step (1), whereinthe substance is a cytokine.
 5. The method of claim 1, whereinantigen-presenting cells are added into the liquid cell culture mediumin the three-dimensional cell culture device co-culture of step (1). 6.A cell population with anti-tumor immune response activity, prepared bythe method of claim 1 wherein the number of cells in the population isfrom 10⁵ to 10¹¹.
 7. A kit, wherein the kit includes: a container, and acell population with anti-tumor immune response activity in thecontainer, prepared by the method of claim 1, wherein the number ofcells in the population is from 10⁵ to 10¹¹.
 8. A method for thetreatment of a patient having a tumor, wherein the method includes thestep of injecting an infusion solution that contains the cell populationwith anti-tumor immune response activity prepared by the method of claim1 into a cancer patient in need of the treatment, wherein themononuclear cells are derived from the patient.
 9. A pharmaceuticalcomposition for active specific immunotherapy for administration to apatient in need of the treatment, comprising using the cell populationwith anti-tumor immune response activity obtained by the method of claim1, wherein the mononuclear cells in the co-culture are derived from thepatient.